Anti-Atherogenic Effects of Vaspin on Human Aortic Smooth Muscle Cell/Macrophage Responses and Hyperlipidemic Mouse Plaque Phenotype

Vaspin (visceral adipose tissue-derived serine protease inhibitor) was recently identified as a novel adipocytokine with insulin-sensitizing effects. Serum vaspin levels are reported either increased or decreased in patients with coronary artery disease. Our translational research was performed to evaluate the expression of vaspin in human coronary atherosclerotic lesions, and its effects on atherogenic responses in human macrophages and human aortic smooth muscle cells (HASMC), as well as aortic atherosclerotic lesion development in spontaneously hyperlipidemic Apoe−/− mice, an animal model of atherosclerosis. Vaspin was expressed at high levels in macrophages/vascular smooth muscle cells (VSMCs) within human coronary atheromatous plaques. Vaspin significantly suppressed inflammatory phenotypes with nuclear factor κB down-regulation in human macrophages. Vaspin significantly suppressed oxidized low-density lipoprotein-induced foam cell formation with CD36 and acyl-coenzyme A: cholesterol acyltransferase-1 down-regulation and ATP-binding cassette transporters A1 and G1, and scavenger receptor class B type 1 up-regulation in human macrophages. Vaspin significantly suppressed angiotensin II-induced migration and proliferation with ERK1/2 and JNK down-regulation, and increased collagen production with phosphoinositide 3-kinase and Akt up-regulation in HASMCs. Chronic infusion of vaspin into Apoe−/− mice significantly suppressed the development of aortic atherosclerotic lesions, with significant reductions of intraplaque inflammation and the macrophage/VSMC ratio, a marker of plaque instability. Our study indicates that vaspin prevents atherosclerotic plaque formation and instability, and may serve as a novel therapeutic target in atherosclerotic cardiovascular diseases.


Introduction
Atherosclerosis is a chronic inflammatory disease arising from endothelial injury and accumulation of cholesterol-laden macrophage foam cells in the artery wall [1]. Macrophages play a key role in vascular inflammation by changing phenotype of pro-inflammatory (M1) or anti-inflammatory (M2) [2].

Expression of Vaspin in Human Vascular Cells
SERPINA12 and vaspin was abundantly expressed in THP1 monocytes, their derived macrophages, HASMCs, human umbilical vein endothelial cells (HUVECs), and human aortic endothelial cells (HAECs) at mRNA and protein expression levels ( Figure 2A).

Effects of Vaspin on Inflammatory Phenotype in Human Monocytes/Macrophages
After 1-6 days of culture, the differentiation of THP1 monocytes into macrophages was confirmed by increased expression of CD68, a macrophage differentiation marker ( Figure 2B). Vaspin (10 ng/mL) did not affect monocyte differentiation into macrophages. However, vaspin (10 ng/mL) significantly decreased the expression of MARCO, an M1 marker, and increased arginase 1, an M2 marker, through differentiation (p < 0.05 to p < 0.0005; Figure 2B). These observations indicated that vaspin shifted the macrophage phenotype overwhelmingly to M2 rather than M1, which was associated with significant changes of nuclear factor κB (NFκB) down-regulation and peroxisome proliferator-activated receptor γ (PPARγ) up-regulation (p < 0.05 to p < 0.0005; Figure 2B).

Effects of Vaspin on ECM Expression in HASMCs
Vaspin significantly increased protein expression of collagen 1 and collagen 3 in HASMCs (both p < 0.05; Figure 5A,B). However, vaspin had no significant effects on protein expression of fibronectin, elastin, and MMP2 in HASMCs (p > 0.05; Figure 5C-E).

Effects of Vaspin on Atherosclerotic Lesion Development in Apoe −/− Mice
Body weight significantly increased with age (17 to 21 weeks old), but did not differ significantly among 21-week-old Apoe −/− mice infused with 3 doses of vaspin (Table 1). There were no significant differences in food intake, systolic and diastolic blood pressures, or plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, and non-HDL cholesterol among four groups of Apoe −/− mice (Table 1). High-dose of vaspin significantly decreased free fatty acid level and tended to decrease fasting glucose and triglyceride levels and homeostasis model assessment of insulin resistance (HOMA-IR) in Apoe −/− mice (Table 1). Low-dose of vaspin significantly increased both plasma insulin level and HOMA-IR in Apoe −/− mice (Table 1).
Both the entire atherosclerotic lesions in aortic lumen surface and plaque size of aortic sinus wall significantly increased with age in Apoe −/− mice (p < 0.0001, p < 0.001; Figure 6A(a,b,e,f),B,C). These were accompanied with significant increases in monocyte-macrophage infiltration, VSMC content, vascular inflammation (pentraxin 3), and the ratio of monocyte-macrophage content/VSMC content in atheromatous plaques (p < 0.05 to p < 0.0001; Figure 6A(i,j,m,n,q,r),D-G). However, 4-week infusion of high-dose vaspin significantly decreased the entire atherosclerotic lesions in aortic lumen surface and plaque size of aortic sinus wall (p < 0.005, p < 0.05; Figure 6A(b,d,f,h),B,C). Vaspin infusion at high dose or both doses significantly decreased monocyte-macrophage infiltration, VSMC content, pentraxin 3 expression, and the ratio of monocyte-macrophage content/VSMC content, a biomarker of plaque instability, in atheromatous plaques (p < 0.05 to p < 0.01; Figure 6A(j-l,n-p,r-t),D-G).

Discussion
The present study provides comprehensive evidence that vaspin inhibits atherogenesis by suppressing the inflammatory phenotype and foam cell formation in macrophages, as well as the migration and proliferation of VSMCs (Figure 7). Vaspin also contributes to stabilizing plaque by increasing collagens and reducing the macrophage/VSMC ratio in atheromatous plaques (Figure 7). Other studies have shown that vaspin inhibits the expression of pro-inflammatory molecules in ECs and monocyte-EC adhesion [16,17]. Lin et al. have shown that vaspin attenuates atherogenesis by inhibiting endoplasmic reticulum (ER) stress-induced macrophage apoptosis in Apoe −/− mice [22].
Vaspin transgenic mice prevented diet-induced obesity, glucose intolerance, and hepatic steatosis, while vaspin-deficient mice developed glucose intolerance associated with up-regulation of ER stress markers [21]. Vaspin improved ER stress and insulin resistance in obese mice by acting as a ligand for cell-surface glucose regulated protein 78/murine tumor cell DnaJ-like protein 1 complex via p-Akt in the liver [21]. Adenovirus vector carrying the full length of the vaspin suppressed neointimal hyperplasia of balloon-injured carotid arteries in streptozotocin-induced diabetic Wistar rats [23]. The intimal proliferation was also suppressed in cuff-injured femoral arteries in vaspin transgenic mice [23]. Several studies have shown that vaspin increases insulin secretion and ameliorates insulin resistance [7,32]. Vaspin also inhibits kallikrein 7-mediated insulin degradation by a classical serpin mechanism [33]. The present study showed that chronic infusion of high-dosing rate of vaspin (5 µg/kg/h) improved insulin resistance and decreased fasting plasma glucose level in Apoe −/− mice. However, chronic infusion of low-dosing rate of vaspin (2.5 µg/kg/h) did not improve insulin resistance with increased fasting plasma glucose and insulin levels in Apoe −/− mice. We speculate that the reason for this is as follows: vaspin infusion into Apoe −/− mice may start to stimulate insulin secretin from pancreas at low doses and accelerate to ameliorate insulin sensitivity in skeletal muscle, fat, and liver in dose-dependent manner. Further studies, such as glucose/insulin tolerance test, are needed to clarify the precise mechanism.
Previous studies have shown that vaspin protects blood vessels by suppressing ROS generation and inflammation via down-regulating the NFκB pathway, and by inhibiting apoptosis via up-regulating the PI3K-Akt-endothelial nitric oxide synthase pathway in ECs [15,[33][34][35]. In the present study, vaspin decreases M1 phenotype acquisition and increases the expression of the M2 phenotype associated with NFκB down-regulation and PPARγ up-regulation in monocyte-derived macrophages.
Vaspin suppresses foam cell formation associated with CD36 and ACAT1 down-regulation, as well as ABCA1, ABCG1, and SRB1 up-regulation in macrophages. Vaspin suppresses the migration and proliferation via the down-regulation of ERK1/2 and JNK pathways, and increases collagen production via the up-regulation of the PI3K-Akt pathway in VSMCs. It is possible that vaspin may not induce apoptosis via activating the PI3K-Akt-Bcl2 pathway in VSMCs.
We discuss the integrity of vaspin levels in our experiments. Several studies have shown that plasma concentrations of vaspin are~2.18 ng/mL in healthy subjects and~0.47 ng/mL in CAD patients [29]. The concentrations of vaspin required for modulation of several responses of THP1 monocyte-derived macrophages and HASMCs were 10-2000 ng/mL in our study, and were 100-150 ng/mL for macrophage and EC responses in other studies [17,20,36]. According to our previous studies [37][38][39], atheroprotective agents are increased to counteract the development of atherosclerosis. The local levels of vasoactive agents could increase to a similar degree by the generation from vascular cells in an autocrine/paracrine fashion [40,41]. Our study shows that the expression of vaspin is increased in macrophages and VSMCs within coronary atherosclerotic lesions. However, decreased circulating blood levels of vaspin in CAD patients may be attributed to severe endothelial dysfunction due to CAD. In monocyte-derived macrophages, the adequate concentrations of vaspin differed in inducing foam cell formation and related protein expression (10-500 ng/mL). This is mostly dependent on the difference in the presence or absence of oxidized LDL. Vaspin at higher than adequate concentrations might lead to the down-regulation of the receptor and intracellular signals. The adequate concentrations of vaspin also differ between macrophages and VSMCs.
Several clinical studies have shown that circulating blood levels of vaspin are significantly increased to improve insulin resistance in patients with type 2 diabetes and metabolic syndrome [24][25][26], but decreased due to severe endothelial dysfunction in patients with CAD [11,29,30]. In patients with type 2 diabetes, vaspin levels are decreased by improvement in insulin resistance with exercise and metformin in men and women, respectively [42,43]. Vaspin levels are increased by rosuvastatin in obese patients with CAD [44]. This is attributed to the improvement effect of rosuvastatin on endothelial dysfunction in CAD patients [45].
In conclusion, the results from the present study indicate that vaspin inhibits atherogenesis by suppressing vascular inflammation, macrophage foam cell formation, and VSMC migration and proliferation. Vaspin also contributes to stabilizing plaque by increasing collagens and reducing the intraplaque macrophage/VSMC ratio. Targeting vaspin allows us to open a therapeutic window for combating atherosclerosis and related diseases, as well as for maintaining vascular health.

Materials
Recombinant human vaspin produced in E. coli was purchased from PeproTech (Rocky Hill, NJ, USA) for in vitro experiments and GenScript (Piscataway, NJ, USA) for in vivo experiments; the purity was ≥98% and >95%, respectively. Rabbit polyclonal anti-human vaspin antibody was purchased from GeneTex (Irvine, CA, USA). Angiotensin II was purchased from Sigma (St. Louis, MO, USA), and phorbol 12-myristate 13-acetate was from Wako (Osaka, Japan). HUVECs and HASMCs were purchased from Lonza (Walkersville, MD, USA) and THP1 monocytes were from Health Science Research Resources Bank (Osaka, Japan).

Human Coronary Artery Immunostaining
This study was deemed exempt as retrospective autopsied coronary artery specimens by the National Cerebral and Cardiovascular Center Review Board (M18-020, 27 July 2006). Written informed consent to autopsy was obtained from families. Buffered 10% formalin-fixed paraffin-embedded human coronary artery specimens from archive autopsy collections of the National Cerebral and Cardiovascular Center were used for immunohistochemistry. Serial cross-sections of coronary arteries from four male patients with CAD (aged 71-87) and three male patients with dilated cardiomyopathy (as non-CAD examples) (aged  were stained with rabbit polyclonal anti-human vaspin antibody. Immunodetection (as a secondary antibody) was performed with the Bond Polymer Refine Detection kit (Leica Biosystems, Newcastle, UK) [37].

Administration of Vaspin into Mice
Animal experiments were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals, with protocols approved by the Institutional Animal Care and Use Committee of Tokyo University of Pharmacy and Life Sciences (No. L15-08, 18 May 2015). A total of 25 male Apoe −/− mice (BALB/c. KOR/StmSlc-Apoe shl mice) at the age of 9 weeks were purchased from Japan SLC and maintained on a normal diet until 13 weeks of age. Subsequently these mice were fed a high-cholesterol diet containing 1.25% cholesterol, 3.0% lard, and 1.625% glucose (Oriental Yeast, Tokyo, Japan). At 17 weeks of age, 7 mice were sacrificed as pre-infusion controls. The remaining 18 were divided into 3 groups of 7, 5, and 6 mice, which were continuously infused with 3 doses of vaspin (0, 50, 100 µg/mouse, respectively) for a period of 4 weeks using osmotic mini-pumps (Alzet Model 1002; Durect, Cupertino, CA, USA). The average dosing rate of continuous infusion was calculated as 0, 2.5, 5 µg/kg/h, respectively. Once every 2 weeks, the mini-pumps were implanted subcutaneously into the dorsum under medetomidine-midazolam-butorphanol anesthesia [48].

Statistical Analysis
All values are expressed as means ± SEM. The data were compared by the unpaired Student's t test between 2 groups and 1-way analysis of variance followed by Bonferroni's post hoc test among ≥3 groups using Statview-J 5.0 (SAS Institute, Cary, NC, USA). Statistical significance was defined as p < 0.05. Funding: This work was supported in part by a Grant-in-Aid for Young Scientists (B) (25860418 to K.Sato), Grants-in-Aid for Scientific Research (C) (16K08943 to K.Sato and 17K08993 to T.W.) from the Japan Society for the Promotion of Science (JSPS), and a Grant-in-Aid for JSPS Fellows (DC2, 17J02716 to R.S.). assignment of human blood plasma for LDL isolation (No. 25J0089, 24 January 2014). Presented in part at the 50th Annual Scientific Meeting of the Japan Atherosclerosis Society, Osaka, Japan, 12-14 July 2018.